Rho proteins are GTP/GDP binding GTPases that belong to the Ras superfamily and are intimately involved in diverse cellular processes and diseases (Symons, M. and Rusk, N. Curr Biol, 2003, 13:R409-418). For example, Rho proteins are pivotal in the regulation of actin cytoskeleton processes such as lamillopodia, fiber and membrane ruffle formation (Fukata, M. et al. Curr Opin Cell Biol, 2003, 15:590-597; Nobes, C. D. and Hall, A. Cell, 1995, 81:53-62). Rho proteins also regulate signal transduction proteins such as Erks, p38 and SAPK, that are involved in the mitogen- and stress-activated kinase pathways (Chang, F. et al. Leukemia, 2003, 17:1263-1293). Most important is the involvement of Rho GTPases as mediators of proliferation and malignant transformation. For example, RhoA and Rac1 are critical for the G1/S cell division cycle traverse (Welsh, C. F. Breast Cancer Res Treat, 2004, 84:33-42), and mediate oncogenic Ras malignant transformation (Welsh, C. F. Breast Cancer Res Treat, 2004, 84:33-42; Downward, J. Nat Rev Cancer, 2003, 3:11-22). In cellular and animal models Rho proteins such as RhoA, Rac1, cdc42 and RhoC have been implicated in invasion and metastasis, and RhoC has been shown to contribute to metastasis in clinical settings (Welsh, C. F. Breast Cancer Res Treat, 2004, 84:33-42; Downward, J. Nat Rev Cancer, 2003, 3:11-22; Clark, E. A. et al. Nature, 2000, 406:532-535).
While most Rho proteins are involved in promoting oncogenesis, invasion and/or metastasis, mounting evidence points to a tumor suppressive role for RhoB. First, in cultured cells, RhoB inhibits oncogenic signaling (Chen, Z. et al. J Biol Chem, 2000, 275:17974-17978; Fritz, G. and Kaina, B. J Biol Chem, 2001, 276:3115-3122), and anchorage-dependent and -independent tumor cell growth (Chen, Z. et al. J Biol Chem, 2000, 275:17974-17978) and induces apoptosis (Chen, Z. et al. J Biol Chem, 2000, 275:17974-17978; Liu, A. et al. Mol Cell Biol, 2000, 20:6105-6113). Second, ectopic expression of RhoB suppresses the growth of human cancer cells in nude mice (Chen, Z. et al. J Biol Chem, 2000, 275:17974-17978; Jiang, K. et al. Mol Cell Biol, 2004, 24:5565-5576). Third, RhoB knockout mice are more sensitive to chemically-induced tumors (Liu, A. X. et al. Mol Cell Biol, 2001, 21:6906-6912) and RhoB (−/−) cells are resistant to apoptosis induced by radiation and cytotoxic agents (Liu, A. et al. Proc Natl Acad Sci USA, 2001, 98:6192-6197). Fourth, ectopic expression of RhoB suppresses EGFR, ErbB2, Ras, PI3K and Akt induced tumor survival, proliferation, invasion and metastasis (Jiang, K. et al. Mol Cell Biol, 2004, 24:5565-5576; Jiang, K. et al. Oncogene, 2004, 23:1136-1145). Fifth, many oncogenes such as EGFR, Ras and Akt suppress the expression of RhoB (Jiang, K. et al. Mol Cell Biol, 2004, 24:5565-5576; Jiang, K. et al. Oncogene, 2004, 23:1136-1145). Finally, in patients with head and neck, lung and brain cancers, RhoB protein levels are drastically decreased as the tumors become more aggressive and highly invasive (Adnane, J. et al. Clin Cancer Res, 2002, 8:2225-2232; Mazieres, J. et al. Clin Cancer Res, 2004, 10:2742-2750; Forget, M. A. et al. Clin Exp Metastasis, 2002, 19:9-15). The above studies suggest that RhoB plays a critical role in suppressing malignant transformation by blocking oncogenic and tumor survival pathways, and that oncogenes such as Ras and EGFR suppress RhoB expression as a step towards malignant transformation.
The fact that RhoA and RhoB have opposing effects on malignant transformation is intriguing considering that RhoA and RhoB share 86% amino acid identity. Presently, it is not understood why RhoA promotes, whereas RhoB suppresses, malignant transformation. The present inventor has carried out site-directed mutagenesis studies with the goal of identifying those amino acids in RhoB that are critical to its tumor suppressive activity.